The most severe consequence of the aging brain is dementia. The number of elderly people is increasing rapidly within our society and as a consequence, dementia is growing into a major health problem. It has been estimated that 25% of the population over the age of 65 have a form of dementia (1) and that the cumulative incidence of dementia in individuals living to the age of 95 is greater than 80% (2,3).
The clinical manifestation of dementia can result from neurodegeneration (e.g. Senile Dementia of the Alzheimer's Type (SDAT), dementia with Lewy bodies (DLB) and frontotemporal lobe dementia (FTLD)), vascular (e.g. multi-infarct dementia) or anoxic event (e.g. cardiac arrest), trauma to the brain (e.g. dementia pugilistica [boxer's dementia]), or exposure to an infectious (e.g. Creutzfeldt-Jakob Disease) or toxic agent (e.g. alcohol-induced dementia) (4). The fact that dementia can result from multiple diseases indicates that the biochemical mechanism(s) of dementia are separate and distinct from the individual disease pathologies. The metabolic basis for the specific cognitive impairments caused by or associated with these specific pathologies are currently unknown.
The differential diagnosis of the types and causes of dementia is not straightforward. A prospective study on the prevalence of SDAT in people over the age of 85 indicated more than half of the individuals with neuropathological criteria for SDAT were either non-demented or were incorrectly diagnosed with vascular dementia. As well, 35% of the clinically diagnosis SDAT subjects did not exhibit neuropathological features sufficient to support the diagnosis (5). Clearly, SDAT symptomology can arise from multiple pathological states that are often clinically indistinguishable. Therefore there is a tremendous need for non-invasive biochemical testing procedures that can accurately identify subjects with a particular neuropathology or increased risk of acquiring a specific neuropathology. SDAT is the most common type of dementia and the percentage of dementias that is SDAT increases with increasing age (2) making this form of dementia the most important one to be able to diagnose in living subjects accurately.
The diagnosis of SDAT ultimately requires demonstration of SDAT pathology, namely the presence of argyrophilic plaques (amyloid deposition) and neurofibrillary degeneration of neurons in the cortex and hippocampus. However, SDAT pathology is often found in the brains of older persons without dementia or mild cognitive impairment (MCI) and may be related to subtle changes in episodic memory (6, 7, 8). At this time, the best post-mortem correlate with dementia in Alzheimer's disease (AD) remains the selective loss/dysfunction of cholinergic projections from the N. basalis and septum to the cortex and hippocampus, respectively.
In SDAT, the cholinergic deficit is best reflected by up to 80% decreases in choline acetyltransferase (ChAT) activity in the neocortex and hippocampus (9, 10, 11, 12, 13). Data indicate that degeneration or dysfunction of cholinergic neurons in the basal forebrain is a defining characteristic of SDAT. Reductions in cortical ChAT activity, monitored via biopsy or in autopsy samples, correlate with the extent of intellectual impairment in SDAT patients, as monitored by the Mini-Mental State Examination (MMSE), an index of global cognitive function (9, 14, 15). In addition, these cortical cholinergic deficits have been found in patients examined within a year of onset of symptoms and cholinesterase inhibitors, which potentiate residual cholinergic transmission, slow the decline in executive memory functions in SDAT patients (16).
Detailed analyses revealed that cholinergic neurons were generally shrunken and dysfunctional, hut not dead, except in late stage AD (13, 18-22). These neuronal phenotypic changes without frank neuronal degeneration occur early in cognitive decline (23). The persistence of shrunken basal forebrain cholinergic neurons in SDAT is similar to that seen in experimental studies of retrograde cellular degeneration in the N. basalis following axotomy (19). It is the applicant's hypothesis that due to the preservation of these magnocellular cholinergic neurons in shrunken form and the applicant's novel discovery of a systemic depletion of key ether lipid molecules that the cholinergic dysfunction in SDAT may be responsive to restorative therapy through pharmacological or supplementation strategies involving ether lipids.
Studies of ChAT levels in the N. basalis and cortex in the same autopsy samples have shown that in 50% of AD patients there is a marked loss of cortical ChAT with no reduction in N. basalis ChAT (13) suggesting abnormal axonal transport in SDAT. In this regard there are significant reductions in frontal (11.9%) and temporal (29.4%) white matter in SDAT autopsy samples compared to normal controls (24). Atrophy of the corpus callosum also is correlated with frontal executive dysfunction in AD patients (25). These observations have led to suggestions that white matter degeneration is an intrinsic component of SDAT (26, 27). Moreover, white matter losses in preclinical SDAT where cortical atrophy is not evident (28), indicate that axonal dysfunction precedes the cortical atrophy observed in clinically manifested SDAT. In fact, white matter lesions are prevalent in aging, in MCI and in early-stage SDAT prior to the development of dementia (29, 30). Again it is this information in combination with the applicant's novel discovery of a systemic depletion of key ether lipid molecules that has led the applicant to the novel hypothesis that the early white matter losses described is due to decreased synthesis of key ether lipid molecules and that this loss could be restored through supplementation of ether lipid molecules.
Lipids make up over 50% of the dry weight of the human brain. Of these lipids, over 60 mol % are phospholipids, and of these phospholipids over 60% are phosphatidylethanolamine (PtdEt or PE) lipids. Ethanolamine phospholipids can be further differentiated based on their sn-1 configurations (either acyl, ether, or vinyl ether). The sn-2 position is typically acyl and the sn-3 position contains the phosphoethanolamine moiety. Therefore the three classes are described as either diacyl (PtdEt), alkyl-acyl (plasmanyl) or alkenyl-acyl (EtnPl). Forty to forty five percent of the ethanolamine phospholipid content is of the PtdEt type and 40-45% is of the EtnPl type, and 10-15% of the plasmanyl type (36).
In the central nervous system (CNS), EtnPls constitute over 80% of the PE content in non-neuronal brain membranes and over 60 mol % in neurons and synaptosomes (34). EntPls in white matter counterparts contain predominantly 18 carbon mono- and di-unsaturated fatty acids (oleic acid (OA, 18:1), linoleic acid (LA, 18:2) at sn-2; in contrast, EtnPls in gray matter contain predominantly longer chain polyunsaturated fatty acids (for example, arachidonic acid (AA, 20:4) and docosohexaenoic acid (DHA, 22:6)) (34). These differences result in different structural characteristics. A high percentage of 18:1/18:2 at sn-2 results in very compact and stable membrane conformations (40, 41), which is consistent with myelin sheath function, whereas a high percentage of AA and DHA results in the fluid membrane structure required for membrane fusion, transmembrane protein function, and intra-cellular-extracellular cholesterol trafficking.
The second critical role that EtnPls play in the CNS is as a key membrane antioxidant. The EtnPl vinyl ether bond acts is preferentially oxidized to form a saturated aldehyde and a 1-lyso, 2-acyl GPE. The preferential oxidation of the vinyl ether linkage preserves sn-2 fatty acids such as DHA and AA (42) that require essential dietary omega-3 and -6 fatty acids, whereas the O-alkyl ether can be re-synthesized in the cell. Oxidation of the vinyl ether bond, however, results in the irreversible turn-over of EtnPls that can only be restored through the re-synthesis of these ether lipids in the peroxisome.
The key point in plasmalogen biosynthesis is that the creation of the 1-O-alkyl bond occurs exclusively in peroxisomes by the enzyme alkyl-dihydroxy acetone phosphate (DHAP) synthase. Loss of function of this enzyme either through point mutations or due to general peroxisomal dysfunction results in a severe plasmalogen deficiency. The remaining key synthetic processes occur in the endoplasmic reticulum (ER) where the sn-2 position is acylated and phosphoethanolamine is added to the sn-3 position to create plasmanyl PE. The final step involves a plasmanyl-specific enzyme that desaturates the 1-O-alkyl ether to form EtnPl.
Pathologically, the formation of extracellular Aβ plaques is a hallmark of SDAT. At the biochemical level, detailed analyses of brain lipids have demonstrated a dramatic (40 mol %) decrease in EtnPl levels of white matter in early SDAT patients, with no further progression in these lipid losses (33-35). In contrast, there is a 10 mol % decrease in gray matter EtnPls in early SDAT which progresses to 30 mol % later in the disease process (34, 35).
Decreased levels of DHA and AA containing EtnPls in gray matter correlate with both dementia severity and Aβ load, however significant changes do not occur until the moderate stage of dementia (34). In contrast, significantly decreased levels of oleic acid (OA)- and linoleic acid (LA)-containing EtnPl in white matter are observed at all stages of dementia (CDR0.5-3.0) in all brain regions, which is consistent with the prevalence of white matter lesions in aging, MCI and pre-dementia SDAT (29, 30). This information supports the present invention that these CNS decreases are the result of a peripheral dysfunction in ether lipid synthesis and not entirely due to oxidative breakdown.
Direct incubation of oligodendrocytes with Aβ peptides selectively decreases plasmenyl PE content (45) and CNS plasmenyl PE decreases correlate with both the temporal and anatomical characteristics of Aβ accumulation animal models (33, 34, 46). Aβ accumulation is also known to directly induce oxidative stress (47-49) and oxidative stress can directly disrupt vesicular fusion, acetylcholine release, and synaptosomal PE content (50). Oxidative stress also preferentially oxidizes EtnPls vs. PtlEts (42, 51). Due to the sensitivity of EtnPls to oxidation, previous researchers have concluded that decreased EtnPls in SDAT tissue is due to increased oxidative stress. It is only through applicant's discovery that both EtnPls and plasmanyl PEs decrease in SDAT that this generally accepted theory is likely to be wrong. To the applicant's best knowledge, this is the first evidence that a systemic reduction in ether lipid synthesis is a causative factor in SDAT.
In both humans and in animal models of Aβ over-production, an age-related trigger is required before these peptides begin to accumulate extracellularly as plaques. In humans, signs of Aβ accumulation start as early as age 40 in non-demented subjects and the prevalence increases with increasing age (53, 54). In mice, genetic conditions that produce 30 times the normal amount Aβ, still fail to result in accumulation until after 8 months of age; thereafter, Aβ begins to accumulate at an exponential rate and preferentially in cortex and hippocampus vs. cerebellum (55). Other animal models of Aβ accumulation show similar age profiles (56). Clearly, Aβ production and accumulation are separately regulated biological processes. The sporadic accumulation of Aβ peptides in SDAT has been linked to a disruption in normal APP processing due to increased membrane cholesterol levels (57). This is consistent with the fact that membrane cholesterol increases with age in both rats and humans (58) and that a high cholesterol diet can increase deposition of Aβ (59).
Although, peroxisomal function as a whole is known to decline with age 6 and appears to be critical for neuronal migration (69), the applicant is the first to link the timing of Aβ accumulation and increased lipid peroxidation (55) to decreased peroxisomal activity in mice (70). Peroxisomal proliferation can inhibit Aβ induced neurodegeneration (71) and preserve cognition in early SDAT (72). Peroxisomes consume between 10 and 30% of total cellular oxygen and generate over 30% of the H2O2. Catalase, the principal peroxisomal enzyme responsible for detoxifying H2O2, decreases in activity with age (73-75), and has been linked with increased lipid peroxidation (73). The decrease in catalase import and increased intracellular H2O, has been linked to severely compromised peroxisomal targeting signals, as age increases (79). Decreased peroxisomal function leads to decreased synthesis of EtnPls and DHA (80, 81), two critical components of normal neuronal functioning, and to increased oxidative stress (79).
With respect to the membrane dysfunction and SDAT, there are well documented age-related decreases in the bioactivities of peroxisomal enzymes involved in the synthesis of plasmalogens and DHA. The two most abundant fatty acids at the sn-2 position of EtnPls in neurons are AA and DHA. AA is an n-6 fatty acid, derived from linoleic acid (18:2, n-6), whereas DHA is an n-3 fatty acid, derived from linolenic acid (18:3, n-3). DHA synthesis involves chain elongation and desaturation of 18:3 n-3 in the ER to 24:6 n-3 with the final step being β-oxidation to DHA in the peroxisome (76). Both DHAP synthase (77) and β-oxidase (75) exhibit decreased function with age. AA synthesis does not require peroxisomal β-oxidation, DHA- and AA-containing EtriPls are selectively decreased with age with DHA-EtnPls being decreased to a greater extent than AA-EtnPls (78).
While the brain contains all of the peroxisomal machinery to synthesize both DHA and EtnPls, studies have shown that DHA is produced and incorporated into phospholipids in the liver, then transported to the brain in this form via the serum. Only trace levels of newly synthesized DHA are found as free fatty acid or in triglycerides (82). This provides further evidence to support the applicant's hypothesis that decreased CNS EtnPls is due to a peripheral dysfunction in ether lipid synthesis and that supplementation of ether lipids will have a positive effect on CNS neuronal composition and function, especially in subjects shown to be deficient in these molecules.
There is thus a need for a diagnostic assay that exploits the biochemical alterations present in SDAT. There is also a need to treat subjects identified as having this biochemical alteration in such a way as to restore this biochemical alteration to normal levels. There is also a need to be able to make this identification as early as possible in the disease progression process as to have maximal benefit to the health of an individual at risk or in the early stages of SDAT.